Chewing lice of Bearded Reedling (Panurus biarmicus) and diversity of louse-host associations of birds in reed beds in Slovakia

A total of 1,621 wild birds representing 34 species were examined for chewing lice in reed beds in southwestern Slovakia during the pre-breeding migration 2008–2009 and 2016–2019. A total of 377 (23.3%) birds representing 15 species were parasitized by 26 species of chewing lice of 12 genera. Dominant genera were Penenirmus (with dominance 32.6%) and Menacanthus (29.4%), followed by Brueelia (12.6%), Acronirmus (10.8%), Philopterus (7.7%), and Myrsidea (4.2%). We evaluated 33 host-louse associations including both 1) host-generalist, parasitizing more than one host species and host-specific lice, occurring only on a single host species, and 2) lice species with large range geographic distribution, reported across the range of the distribution of their hosts and lice species with only occasional records from a limited area within the range of their hosts. The Bearded Reedling, Panurus biarmicus (Linnaeus, 1758), was parasitized by two species of chewing lice, Menacanthus brelihi Balát, 1981 and Penenirmus visendus (Złotorzycka, 1964), with conspicuously different prevalences (5.6% vs. 58.2%, respectively; n = 251). New material enabled us to redescribe both species of lice: the first one is resurrected from previous synonymy as a valid species. A fragment of the mitochondrial cytochrome oxidase I gene was sequenced from these two species in order to assess their relative phylogenetic position within their genera. Our study demonstrates the importance of an adequate identification of parasites, especially on rarely examined and endangered hosts.


Introduction
A diverse assemblage of chewing lice (Phthiraptera) and their host associations are well-known from Slovakia.Recently we reviewed all available published records, old museum collections, and recently collected material of chewing lice from Slovakia and provided a checklist containing 249 species of chewing lice -65 amblyceran species from 22 genera representing the families Laemobothriidae, Menoponidae, and Ricinidae, and 184 ischnoceran species from 54 genera of the family Philopteridaeand 358 host-louse associations from 171 bird species from 21 orders [36].Nevertheless, information about infestation characteristics like prevalence or mean intensity is still scarce, incomplete, and focused on louse species from hosts occurring at higher elevations, in montane forests and shrublands [2,8,[17][18][19][20].
We recently focused on a lowland wetland bird community.We evaluated infestation characteristics of ectoparasites in relation to the migration behavior and sexual dimorphism of five passerine bird species that represent habitat specialists breeding in this environment dominated by Common Reed (Phragmites australis) [51].In addition to habitat specialists, i.e., Acrocephalus spp., Locustella luscinioides (Savi, 1824), and Panurus biarmicus (Linnaeus, 1758), the avifauna of the reed beds also includes birds that use this environment to roost during both the breeding season and migration, e.g., Hirundo rustica Linnaeus, 1758, Motacilla alba Linnaeus, 1758, and Sturnus vulgaris Linnaeus, 1758 [53].
The Bearded Reedling, Panurus biarmicus, has a wide range across the Palearctic realm.It is evaluated as Least Concern in the Red List Assessment [7].The distribution of the species has seen significant gains elsewhere across its European range [26], but the populations in Slovakia have decreased within the last few years [6] and they are considered as Near Threatened there [10].
We focused on the lice of P. biarmicus, and only two species of chewing lice are known from this host -Menacanthus eurysternus (Burmeister, 1848) and Penenirmus visendus (Złotorzycka, 1964) [40].Menacanthus lice were first reported from P. biarmicus by Balát [4] who described them as Menacanthus brelihi Balát, 1981.Balát [4] included one picture of male parameres and photographs of the holotype female and paratype male, with a short general text description.Consequently, Krištofík [29] examined the Balát's type material and stated that "examined slides are of bad quality".He concluded his re-examination of this material with the statement that all the main features of examined specimens were identical to Me. eurysternus.As a result, he synonymized Me. brelihi with Me. eurysternus.Penenirmus visendus was originally described as Panurinirmus visendus by Złotorzycka [55] based on a single female from the Bearded Reedling from Poland.Later, Emerson [11] synonymized Panurinirmus with Penenirmus.To date, no description of the male of this species has been published.
In this paper, we extend the knowledge of ectoparasites of passerine birds occurring in reed bed ecosystems.The aims of this paper are to: (1) present new data on the species distribution of chewing lice found on birds in reed beds in southwestern Slovakia; (2) clarify information on their infestation characteristics; (3) redescribe both sexes of Penenirmus visendus and Moiuytrewuytreqenacanthus brelihi and resurrect the latter as a valid taxon; and (4) confirm the validity of these taxa also by phylogenetic analysis of a fragment of the mitochondrial cytochrome oxidase I (COI) gene.
The specimens examined are deposited at the Moravian Museum, Brno, Czechia (MMBC), Museum of Natural History, University of Wrocław, Poland (MNHW), and at the Department of Biology and Wildlife Diseases, University of Veterinary Sciences Brno, Czechia (UVSB).
Sequences of a 379 bp fragment of the COI gene were obtained from Menacanthus brelihi and from Penenirmus visendus from Panurus biarmicus using methods described by Johnson et al. [22].New sequences (GenBank accession numbers OR533291-OR533294, OR626644-OR626646) were aligned together with all available sequences from Menacanthus and Penenirmus genera previously published in the literature and deposited in GenBank [21-24, 31-32, 34, 49, 52] using Geneious 9.1.8[25] in order to assess their genetic divergence and interspecific relationships.For phylogenetic analysis, we first computed the Akaike information criterion (AIC) computed in MEGA 7.0.14[30] to identify the most appropriate model of nucleotide substitution.The phylogenetic tree was built with the Bayesian inference analysis (BI) using the Mr. Bayes 3.2.6 plugin in Geneious 9.1.8[25,44] with a GTR + G + I model for 10(7) generations, with trees sampled every 1,000 generations.A majority rule consensus tree was summarized after discarding 1,000 trees as a burn-in.Computation of genetic p-distances was performed in MEGA 7.0.14[30].

Chewing lice of the
Type locality: Velký Dvůr near Pohořelice (south Moravia), Czechia Remarks.Menacanthus brelihi belongs to the curuccae species group (sensu Martinů et al. [32]).Both sexes of this species are readily identified by combination of characters as follows: (1) each side of metanotum with 3-5 lateroanterior setae; (2) characteristic shape of gular plate, with large central lighter "hole" and several small anterior ones, posterior margin straight or undulated, all four setae on each side are inserted in clear lateral area; (3) ocular seta 19 finer than outer central pronotal seta 1; (4) pleurites with anterior setae; (5) large number of sternal setae, especially on sternites III-VI of females (each with more than 53 setae) and sternites III-V of males (each with more than 34 setae); and (6) quite large size.In the key by Price [39], the female of Me. brelihi would key out to couplet 28, being closest to Me. robustus (Kellogg, 1896).However, the female of Me. brelihi can be distinguished from that of Me. robustus by a different number of setae on tergites VII and IX (28-29 and 23-26 vs. 23-26 and 32-33) and sternites IV-V (67-72 and 63-75 vs. 60-65 and 51-56).Although males of Me. robustus are unknown, the male of Me. brelihi would key out to couplet 35 in the key by Price [39], being closest to Me. tenuifrons Blagoveshtchensky, 1940.However, the male of Me. brelihi can be distinguished from that of Me. tenuifrons by a different number of setae on tergite IX (7-8 vs. 10-11) and sternites IV-VII (a total of 114-143 vs. 97-118).
In order to add Me. brelihi to Price's [39] key, the following alterations should be made:    Note: Balát's collection at MMBC includes seven slides with Me. brelihi.According to Balát's notes, all three specimens from the type series were collected from an adult male of P. biarmicus.Although Balát [4] mentioned a total of 11 lice (1#, 3$$ and 7 nymphs) from the adult female of the type host that were examined at Nová Ves, only one slide with one female and one nymph is present at the MMBC.Similarly, according to Balát's notes, specimens from Austria were collected from two females (2$$ from 17 Sep.1960) and one male of P. biarmicus (1#, 4$$ and 2 nymphs from 18 Sep.1960), but only three females had been mounted on the slide, while other specimens were stored in ethanol.Except for the specimens mentioned within the examined material, all other specimens are missing from the MMBC and must be regarded as lost.
Parvorder Type locality: Górki Wschodnie near Gdańsk, Poland Remarks.Złotorzycka [55] provided a very poor description of P. visendus under the name Panurinirmus visendus based on a single female from P. biarmicus from Poland.There is no comprehensive morphological revision of Penenirmus from passerine birds and that is one reason why species determination is difficult and a common practice is to identify species based on host records.To date, 11-14 species of Penenirmus have been reported from Europe [33,40,57].To partially address the difficulties in identifying lice in this genus, we compare P. visendus with P. albiventris a recently welldescribed by Sychra et al. [49].We found only limited morphological differences between these two species concerning mainly slight differences in the shape of the dorsal anterior head plate (with short blunt posterior process in P. visendus compared with quite long and pointed process in P. albiventris) and abdominal tergites II-VI (joined by two narrow conspicuously pigmented strips compared with tergites that are joined by a single pigmented strip).A fragment of the COI gene was sequenced from four specimens of P. visendus (GenBank accession numbers OR533291-OR533292).Comparing our sequences with other available sequences from Penenirmus genus, the closest were those of P. albiventris, with sequence divergences around 18-19%.These sequence divergences are large enough to confirm P. visendus as a separate species, at least until sequences from other species are known.These data support the aforementioned morphological differences.Phylogenetic relationships among sequences obtained from P. visendus and sequences from other Penenirmus species are presented in Figure 7.
Male (n = 20).As in Figures 4A and 6D.Head (Fig. 5A) with postantennal suture, with one post-nodal and three posttemporal setae on each side, all of them short and spine-like.Marginal temporal setae 1 and 3 long, other marginal temporal setae short.Anterior dorsal setae of forehead shorter than distance between them.Dorsal anterior head plate quite large with slightly concave anterior margin and short blunt posterior process (Fig. 5B).Metanotum and metapleurite with an almost continuous row of 7 evenly spaced setae on each side (outmost lateral short metapleural seta included).Mesosternal plate with 2 setae, metasternal plate with 4 setae.

Discussion
The avifauna of the reed beds represents a mixture community of both habitat specialists that breed in this environment, as well as bird species that only roost in reed stands during both the breeding season and migration.In the case of habitat specialists, Sychra et al. [51] found significantly higher prevalences and mean abundances of chewing lice on resident and short-distance migrants (Acrocephalus melanopogon, Panurus biarmicus) than on long-distance migratory species (Acrocephalus scirpaceus, A. schoenobaenus, Locustella luscinioides).Except for the aforementioned birds, the highest infestations of lice in this study were found on birds roosting in reed beds in larger flocks, such as Hirundo rustica or Sturnus vulgaris.Horizontal transmission of lice is more frequent in communal roosting places where close contact between larger number of individuals can take place.Prevalence and intensity of lice are thus usually higher as well on these birds [27,45].We suggest that reed beds play an important role in the maintenance and dispersal of ectoparasites in the population of these hosts.On the other hand, despite large sample sizes, neither lice nor eggs were found on Acrocephalus arundinaceus (n = 107), even though Menacanthus curuccae and Philopterus fedorenkoae (Mey, 1983) are known from this host [35,40].
In the present study, we evaluated 33 host-louse associations including 12 genera of lice.These associations include both 1) host-generalist, parasitizing more than one host species and host-specific lice, occurring only on single host species, and 2) lice species with broad geographic distribution, reported across the range of distribution of their hosts and lice species with only rare records in a limited area (for hosts see Price et al. [40]; for distribution see Mey [33], unless otherwise noted).
Undetermined species of Penenirmus have also been reported from Hungary by Rékási [41].We extend the area of distribution of this louse species to include Austria and Czechia.
In our study, Panurus biarmicus has fragmented distributions in Central Europe.Bush et al. [9] showed that habitat fragmentation may impact the prevalence of lice.The low infestation indices of Menacanthus brelihi may follow a similar scenario, i.e., smaller populations of hosts on the edge of their range may harbor fewer lice [38].Sampling of P. biarmicus in other parts of its range is necessary to confirm that the rareness of this species is related to habitat fragmentation.On the other hand, our results show that Penenirmus visendus is a common parasite of P. biarmicus, occurring with quite high prevalence throughout year [51].Therefore, we suppose that P. visendus could have a stronger impact on its host than Me.brelihi.However, we cannot exclude that P. visendus is, on the contrary, a less virulent parasite that can occur in higher numbers without having a significant effect on the condition of its host.An experimental study is necessary to evaluate these assumptions.The present study also demonstrated the importance of accurate identification of parasites, especially on rarely examined and endangered species, where the knowledge of parasite diversity can be useful in their conservation programs.Moreover, Gustafsson et al. [16] suggested that parasites on endangered hosts, especially those that are host-specific, should also be treated as endangered.Panurus biarmicus is evaluated as Near Threatened in Slovakia [10], so if we adopt the idea of Gustafsson et al. [16], then both louse species on this host, but especially Me. brelihi, may be considered to have the same conservation status.

Figure 3 .
Figure 3. Diagram of the evolution of the Menacanthus lineages.The topology was adapted from the cytochrome oxidase subunit I phylogeny in Supplementary Figure S1.The origin of the samples is in parentheses: AF -Afrotropical realm; EPA -Eastern Palearctic realm; NA -Nearctic realm; NT -Neotropical realm; OR -Oriental realm; WPA -Western Palearctic realm.

Figure 7 .
Figure 7. Phylogenetic tree of the Penenirmus species based on partial COI sequences, estimated with Bayesian analysis based on a 379 bp alignment of a COI gene fragment.The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.The numbers above the branches indicate Bayesian posterior probabilities.Branches with posterior probabilities < 0.5 were collapsed.New sequences are in bold type.The origin of the samples is in parentheses: AF -Afrotropical realm; NA -Nearctic realm; NT -Neotropical realm; OR -Oriental realm; WPA -Western Palearctic realm.

Table 1 .
List of hosts and their chewing lice.P/E = prevalence = number of birds parasitized (P)/number of birds examined (E); MI = mean intensity = number of individuals of a particular chewing louse species on infested hosts; MA = mean abundance = number of individuals of a particular chewing louse species on examined birds; AE = a total number of collected lice; N = nymphs; M = Menoponidae; P = Philopteridae; R = Ricinidae.